Mismatch repair detection

ABSTRACT

Mismatch Repair Detection (MRD), a novel method for DNA-variation detection, utilizes bacteria to detect mismatches by a change in expression of a marker gene. DNA fragments to be screened for variation are cloned into two MRD plasmids, and bacteria are transformed with heteroduplexes of these constructs. Resulting colonies express the marker gene in the absence of a mismatch, and lack expression in the presence of a mismatch. MRD is capable of detecting a single mismatch within 10 kb of DNA. In addition, MRD can analyze many fragments simultaneously, offering a powerful method for high-throughput genotyping and mutation detection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/271,055, flied Mar. 17, 1999 now U.S. Pat. No. 6,406,847, which is acontinuation-in-part of U.S. patent application Ser. No. 08/713,751,filed Sep. 13, 1996 now abandoned; which claims priority to U.S.Provisional Patent Application No. 60/004,664, filed Oct. 2, 1995.

GOVERNMENT GRANTS

This invention was made with government support under Contract Nos. HD24610 07-10 and 5T32 GM07618 awarded by the National Institutes ofHealth. The Government has certain rights in this invention.

BACKGROUND

The detection of mutations in genomic DNA plays a critical role inefforts to elucidate the genetic basis of human disease. For many typesof genetic screening and analysis, knowledge of the presence of amutated copy of a gene is essential. Such information may be used inprenatal and other genetic testing, as well as analysis of tumor cellsand other somatic mutations. For many genes, there are a number ofdifferent mutations that can affect function.

Common diseases such as diabetes, heart disease and psychiatricdisorders are caused in part by genetic variations in multiple genes.Genetic variations are not only involved in the genesis of diseases butthey are also chief determinants of disease progression and response totreatment. Identification of the genetic variations involved in commondiseases can greatly improve the diagnosis, prognosis, and treatment ofsuch diseases.

One approach for identifying the potentially causative variationsinvolved in common diseases is to screen patients and controls forgenetic variations in a large number of candidate genes. Genetic codingsequences constitute less than 5% of the entire human genome, yet thevast majority of human diseases are caused by sequence variation inthese coding sequences. Reagents for large scale screening of genes arealready available, as a significant proportion of human gene sequencesexists in the rapidly expanding public databases. Many DNA variationscreening methods have been developed, e.g. single strandedconformational polymorphism (SSCP); and high performance liquidchromatography (HPLC). Since these methods are not designed to screenmany genes simultaneously, their usefulness has been limited to testinga handful of candidate genes.

In the absence of high throughput technology capable of large scalescreening of genes for the identification of variations involved indiseases, less straight forward approaches such as association andlinkage mapping have been proposed. In these approaches, neutral geneticvariations (polymorphic markers) are cataloged into a genetic map. Thesepolymorphic markers are used in a genetic linkage or associationanalysis to approximate the chromosomal location of the disease genes.

Association studies are based on the probability that certainpolymorphisms in close proximity to the ancestral disease-causingvariation are still present in today's patient population. In linkage orassociation mapping one hopes that at least a single marker issufficiently close to the disease-causing variation, and therefore wouldco-segregate with the disease in a family or in a population. Theanalysis assumes that a large proportion of the mutations had a singlepoint of origin.

Linkage and association based approaches have been successful formapping of simple Mendelian diseases. However, mapping of diseases witha complex mode of inheritance has been less successful. Identificationof the variations that are involved in such diseases is widely believedto require the performance of association analysis using tens ofthousands of markers. Because single nucleotide polymorphisms (SNPs) arethe most prevalent polymorphisms, they are proposed to be the markers ofchoice for these association studies.

Multiple methods, such as chip hybridization and oligonucleotideligation assay (OLA), have been developed for genotyping of SNPs. Allthese SNP genotyping methods operate on a common principle of genotypinga previously identified single base polymorphism. Polymorphic sites arefirst identified by sequencing multiple individuals, then compiled intoa map. Finally, patients and controls are tested for the presence orabsence of each polymorphism.

In view of the importance of genetic testing, methods whereby one caneasily screen for genetic mismatches between two DNA molecules is ofgreat interest. A simple method to determine whether two DNA moleculesare identical or different, and that is capable of multiplex analysiswould be of great benefit in these analyses.

Relevant Literature

Techniques for detection of conformational changes created by DNAsequence variation as alterations in electrophoretic mobility aredescribed in Orita et al. (1989) P.N.A.S. 86:2766; Orita et al. (1989)Genomics 5:874; Myers et al. (1985) N.A.R. 13:3131 (1985); Sheffield etal. P.N.A.S. 86:231; Myers et al. Meth. Enzym 155:501; Perry and Carrell(1992) Clin. Pathol. 45:158; White et al. (1992) Genomics 5:301.Techniques that use chemicals or proteins to detect sites of sequencemismatch in heteroduplex DNA are described in Cotton et al. (1988)P.N.A.S. 85:4397; Myers et al. (1985) Science 230:1242; Marshal et al.(1995) Nature Genetics 9:177 (1995); Youil et al. (1995) P.N.A.S. 92:87.Chip hybridization is described in Wang et al. Science 280: 1077-82.

Grompe (1993) Nature Genetics 5:111 reviews methods for screening largestretches of DNA. Mapping strategies may be found in Risch (1990) Am. J.Hum. Genet. 46:229-241; Lander and Botstein (1987) Science236:1567-1570; and Bishop and Williamson (1990) Am. J. Hum. Genet.46:254-265. Sandra and Ford, (1986) Nucleic Acids Res. 14:7265-7282 andCasna, et al (1986) Nucleic Acids Res. 14:7285-7303 describe genomicanalysis.

However, several approaches are presently available to isolate large DNAfragments, including long range PCR with enzymes with high fidelitydescribed in Nielson et al. (1995) Strategies 8:26; recA-assistedcleavage described by Ferrin and Camerini-Otero (1991) Science 254:1494;and the use of a single set of oligonucleotide primers to PCR amplifymultiple specific fragments simultaneously in Brookes et al. (1995)Human Molecular Genetics 3:2011.

The E. coli methyl mismatch repair system is described in Wagner andMesselson (1976) P.N.A.S. 73:4135; Modrich (1991) Annu. Rev. Genet.25:229; Parker and Marinus (1992) P.N.A.S. 89:1730; and Carraway andMarinus (1993) J. Bacteriology 175:3972. The normal function of the E.coli methyl-directed mismatch repair system is to correct errors innewly synthesized DNA resulting from imperfect DNA replication. Thesystem distinguishes unreplicated from newly replicated DNA by takingadvantage of the fact that methylation of adenine in the sequence GATCoccurs in unreplicated DNA but not in newly synthesized DNA. Mismatchrepair is initiated by the action of three proteins, MutS, MutL andMutH, which lead to nicking of the unmethylated, newly replicated strandat a hemimethylated GATC site. The unmethylated DNA strand is thendigested and resynthesized using the methylated strand as a template.The methyl-directed mismatch repair system can repair single basemismatches and mismatches or loops of up to four nucleotides in length.Loops of five nucleotides and larger are not repaired.

The use of site specific recombinases in eukaryotic cells is describedby Wahl et al., U.S. Pat. No. 5,654,182; and by Sauer, U.S. Pat. No.4,959,317.

SUMMARY OF THE INVENTION

Compositions and methods are provided for an in vivo bacterial assay,termed “Mismatch Repair Detection” (MRD). The method detects mismatchesin a double stranded DNA molecule, where the sequence of one stranddiffers from the sequence of the other strand by as little as a singlenucleotide. The two strands of the DNA molecule are from differentsources. One strand is unmethylated DNA, having a detectable marker geneand the sequence being tested for mismatches. The other strand ismethylated DNA, having an inactivated copy of the marker gene where thedefect does not activate repair mechanisms, and another copy of thesequence to be tested. Heteroduplex dsDNA formed from the hybridizationof the two strands is transformed into a bacterial host with an activemethyl mismatch repair system (MMR host).

The host repair system is activated by a mismatch in the sequence ofinterest, and will then “co-repair” the marker gene, to produce aninactive, double stranded copy. When the two strands of the sequence ofinterest are a perfect match, the marker gene is not altered, and thetransformed bacteria will produce active marker. Where a mismatch ispresent, the transformants are readily identified by the lack of activemarker, and may then be isolated and grown for further analysis. MRD isa rapid method for analysis of numerous fragments simultaneously. It isuseful as an assay for enumerating differences between various sourcesof DNA, and as a means of isolating DNA with variant sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the method for mismatch repair detection.

FIG. 2 depicts the method using single or double stranded vectors and anamplification product as a test sequence.

FIG. 3 shows a plasmid map of pMF200 and pMF100.

FIG. 4 depicts formation of heteroduplex DNA

FIG. 5 depicts analysis of MRD results by hybridization.

FIG. 6 is a schematic of MRD utilizing cre/lox as a detectable marker.

FIG. 7 is an acrylamide gel read-out of screened fragments.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Mismatch Repair Detection (MRD) is a method of detecting mismatches inthe sequence of a double stranded DNA molecule. The method willdetermine whether two DNA sequences differ by as little as a single basechange, in a region of over 10,000 nucleotides. Multiple DNA fragmentscan be analyzed in a single reaction, and the process is easily scaledup to run large numbers of reactions in parallel. Depending on the inputDNA, MRD can be used for various purposes. It is used in genetic mappingby testing a large number of polymorphic markers in order to analyzelarge regions of eukaryotic chromosomes for the presence of mutations.In a large pool of genomic or cDNA clones, the method will identifythose DNAs where there is a mismatch between the control and testpopulation, providing a particularly simple method of isolating variantalleles from a particular locus or region. The method can also be usedto detect somatic changes in DNA, such as those found in tumor cells, orin the hypermutation of antibody genes. A key advantage of MRD is that,once provided with suitable vectors, the procedure is extremely easy toperform.

The ability to perform high throughput DNA variation detection makesMismatch Repair Detection (MRD) ideal for performing association anddirect screening studies. MRD's multiplexing potential exceeds that ofcurrently known methods, therefore offering an improvement over othermethods for large scale SNP genotyping.

MRD also can be used to screen a massive number of candidate genes inorder to identify disease-causing variations. It is possible to test thecoding regions of all human genes in a limited number of MRD reactions.Testing the coding regions of all the genes in a population of patientsand controls will readily reveal disease-causing variations. Sensitivityof this direct approach is significantly higher than that of theassociation studies as it does not require assumptions as to the originof mutation and the prevalence of the disease-carrying ancestralchromosome in the patient population. Methods detecting disease-causingvariations directly are more likely than association methods to succeedin identifying these variations. This direct candidate gene screeningapproach is powerful and effective and can greatly accelerate theidentification of variations causing clinically-significant phenotypes,greatly improving disease diagnosis, prognosis, and treatment.

Applications of the method based on direct screening of disease genesinclude diagnosis; sub-diagnosis where one distinguishes betweenmutations in two related disease associated genes, e.g. factor VIII vs.factor IX deficiency; prognosis of disease susceptibility; treatmentdevelopment; and treatment optimization.

MRD Method

Mismatch Repair Detection (MRD) utilizes Escherchia coli′s ability torecognize mismatches in order to detect DNA variations. Many DNAfragments carrying a potential mismatch can be introduced simultaneouslyinto the same E. coli culture. Each cell functions as a separatemismatch detection entity. Separation of cells that detected mismatchesfrom those that did not provides two pools of cells containingfragments—one pool with and one without mismatches. The complex processof variation detection is then reduced to the relatively simple task ofidentifying the DNA fragment content of each pool.

E. coli detects single point mismatches as well as one-, two-, andthree-nucleotide loops, but it does not detect loops of 5 nucleotides ormore. The template for repair by E. coli is a hemimethylated doublestranded DNA. Mismatches in the hemimethylated DNA activate E. coli′smismatch repair pathway and result in a large portion of theunmethylated strand (if not its entirety) being degraded and themethylated strand serving as a template to be recopied.

MRD exploits the ability of bacterial cells to “co-repair” longstretches of DNA. When the two strands of a dsDNA molecule have amismatch, i.e. the nucleotides at a specific position are notcomplementary, the methyl-directed mismatch repair system of a bacteriawill excise and replace the incorrect nucleotide. The strand of DNA thatcontains within it the modified sequence motif GA^(methyl)TC isrecognized by the repair system as the “correct” sequence. Correction isinitiated by mismatches of one to four contiguous nucleotides. A loop of5 or more mismatched nucleotides is not recognized by the proteinsresponsible for initiation of repair, and will remain uncorrected in theabsence of other mismatches. However, if repair is initiated at one siteon the DNA molecule, then a region extending for at least 10 kb will beco-repaired on the molecule.

The subject method uses a two vector system where each vectorcontributes one strand to the double stranded test vector. Onecontributing vector contains a gene encoding an active, detectablemarker. For convenience, this will be referred to as the “A vector”, orthe “standard”. The second contributing vector is substantiallycomplementary to the A vector, except that the marker gene has aninactivating insertion, deletion or substitution loop of at least about5 nucleotides in length. This vector will be referred to as the “I”vector. The A vector and the I vector may be replicated as doublestranded DNA, which is then denatured to form single strands, or thevectors may be grown as single stranded entities. The A vector will bereplicated under conditions that do not methylate adenine at the GATCrecognition site, whereas the I vector will be modified to havemethylated adenine at these sites.

One strand from the A vector and one strand from the I vector areannealed to form a heteroduplex, double stranded “A/I” vector. The A/Ivector will be methylated on only one strand, e.g. the strand that iscontributed by the I vector. When the A/I vector is transformed into asuitable bacterial host having an active methyl mismatch repair system(MMR host), the loop between the active and inactive marker gene willnot initiate repair. Correction of the marker gene will only take placewhen there is a mismatch capable of initiating repair elsewhere in themolecule.

The A/I vector is ligated to a “test sequence”. The test sequence is adouble stranded DNA molecule comprising the sequence of interest, whichis being tested for mismatches. A mismatch in the test sequence willinitiate repair of the loop in the marker gene in the bacterial hostcell. Each strand of the test sequence is contributed by a differentsource, herein termed X and Y strands. One or both of the X and Ystrands may be ligated to the A and I vectors prior to the previouslydescribed annealing step. In one embodiment of the invention, the Avector is ligated to the test sequence, and replicated as a singlestranded “standard” vector. Alternatively, the X or Y strand(s) isligated to the double stranded A/I vector. The A/I vector ligated to thetest sequence shall be referred to as the test vector.

When the X and Y strands of the test sequence are perfectlycomplementary, then bacteria transformed with the test vector will notinitiate correction of the loop in the marker gene, and will express amixture of the active and inactive marker. If X and Y are mismatched,then repair is initiated. The marker gene will be “corrected” byco-repair, so that both strands will have the inactive marker sequence.Transformed bacteria will therefore lack active marker. The transformedbacteria are grown on plates, liquid culture, etc., under conditionswhere expression of the marker can be detected. The presence oftransformants that lack the marker indicates a mismatch in the testsequence. These transformants may then be isolated for further use. FIG.1 is a schematic depicting this process.

DNA Vectors

The A and I vectors may be any double stranded or single strandedepisomal DNA element that is replicated in the MMR bacterial host, e.g.phage, plasmids, bacterial artificial chromosomes (BACs), etc. Manyvectors are known in the art and are commercially available. The twovectors are substantially complementary if single stranded, andsubstantially identical if double stranded, except for the previouslydiscussed loop in the marker gene, and optionally, the X or Y sequenceof interest. Double stranded vectors must be linearized and denaturedprior to formation of the A/I vector. The vectors will contain at leastone methylation recognition sequence, generally GATC, more usuallymultiple recognition sequences will be present.

The A and I vectors have an origin of replication that is active in theMMR host cell. The origin may provide for a high or low copy number ofthe vector. Optionally, the vectors will include a gene encoding aselectable marker, e.g. antibiotic resistance; genes or operons thatcomplement a metabolic defect of the MMR host; resistance to phageinfection, etc. Phage vectors may include packaging signals, genesencoding phage coat proteins and regulatory genes, etc. Desirably, thevector will contain a polylinker having a number of sites forrestriction endonucleases to facilitate cloning.

The detectable marker gene may be any gene expressed in the bacterialhost that provides a directly or indirectly detectable characteristic.Directly detectable markers of interest include antibiotic resistance,color change of a substrate, expression of luciferase, etc. In oneembodiment of the invention, the marker is a recombinase, e.g. crerecombinase, FLP recombinase, pSR1 recombinase, etc., which isindirectly detected. For example, the presence of active cre may bedetected by recombination between two or more heterologous recombinationsites, where a directly detectable marker is present between theserecombination sites. The active enzyme will recombine between the sites,thereby deleting the directly detectable marker; while in the presenceof inactive enzyme the directly detectable marker is maintained. Suchdirectly detected markers need not be present on the actual vector, andmay be contained on the bacterial chromosome or on another episome.

The term “heterologous recombination site” is meant to encompass anyintroduced genetic sequence that facilitates site-specificrecombination. In general, such sites facilitate recombination byinteraction of a specific enzyme with two such sites. Exemplaryheterologous recombination sites include, but are not necessarilylimited to, lox sequences; recombination mediated by Cre enzyme; frtsequences (Golic et al. (1989) Cell 59:499-509; O'Gorman et al. (1991)Science 251:1351-5; recombination mediated by the FLP recombinase), therecognition sequences for the pSR1 recombinase of Zygosaccharomycesrouxii (Matsuzaki et al. (1990) J. Bacteriol. 172:610-8), and the like.

Sequences encoding lox sites are of particular interest for use in thepresent invention. A lox site is a nucleotide sequence at which the geneproduct of the cre gene, catalyzes site-specific recombination. Aparticularly preferred lox site is a loxP site. The sequence of loxP,which is 34 bp in length, is known and can be produced synthetically orcan be isolated from bacteriophage P1 by methods known in the art (see,e.g. Hoess et al. (1982) Proc. Natl. Acad. Sci. USA 79:3398). The loxPsite is composed of two 13 bp inverted repeats separated by an 8 bpspacer region. The nucleotide sequences of the insert repeats and thespacer region of loxP are as follows: SEQ ID NO:1 ATAACTTCGTATA ATGTATGCTATACGMGTTAT

Other suitable lox sites include loxB, loxL, and loxR, which can beisolated from E. coli (Hoess et al. (1982) Proc. Natl. Acad. Sci. USA22:3398). The nucleotide sequences of the insert repeats and the spacerregion of loxC2 are as follows: SEQ ID NO:2 ACMCTTCGTATA ATGTATGCTATACGAAGTTAT

The heterologous recombination sites useful in the present invention maybe either a naturally-occurring sequence or a modified sequence. Forexample, PCT published application no. WO 93/19172 describes phagevectors in which the VH10 genes are flanked by two loxP sites, one ofwhich is a mutant loxP site. Lox sites can also be produced by a varietyof synthetic techniques which are known in the art. For example,synthetic techniques for producing lox sites are disclosed by Ogilvie etal. (1981) Science, 210: 270.

Other examples of indirectly detected markers include regulatoryfactors, e.g. a repressor in an strain constructed to carry one or moregenes that are regulated by the specific repressor. Another example of agene that can have indirect effect on one or more directly detectedmarkers is the amber suppressor supF (or ochre, or opal suppressor).

The use of markers that provide for a color change may be detected bygrowing the transformed bacteria on medium that allows for the colorchange, but where the active marker is not required for growth.Transformants expressing the marker are then detectable by visualinspection, spectrophotometry, flow cytometry, etc. Another example of adirectly detected marker is a gene that can be expressed on the surfaceof the bacterium and can therefore be detected by antibodies to it. Theuse of antibiotic resistance as a detectable marker, e.g. expression ofβ-lactamase, etc. may require duplicate plates to isolate the mismatchedsequence. Alternatively, an antibiotic resistance and an antibioticsensitivity gene may both be present. For example, the vector maycontain a streptomycin sensitivity and a tetracycline resistance gene.When both active genes are present, then cells may be grown in duplicatecultures, one containing streptomycin, and the other containingtetracycline. In another example, transformants are grown undernon-selective conditions, and a duplicate plate grown under selectiveconditions. The colonies that cannot grow in the presence of theantibiotic have a mismatched test sequence. A convenient marker is theLacZα gene, which permits the induction of β-galactosidase expression inthe presence of isopropyl-β-D-thiogalactoside (see Messing, supra.). Theβ-galactosidase cleaves indolyl-β-D-galactoside to produce a coloredproduct.

The inactivated marker gene on the I vector has an insertion, deletionor substitution “loop” of at least about 5 nt. The minimum size of theloop is required because the loop must not initiate repair by the MMRhost. Larger loops, of as much as several hundred bases, may beintroduced, but are not necessary for the practice of the invention. Theloop inactivates the marker gene by introducing a frameshift, stopcodon, etc.

In most cases, the I vector will provide the methylated strand. This isdone so that during co-repair, the marker gene will be converted to theinactive form. For a number of markers, the active gene is dominant overthe inactive. For example, a transformant containing one activeantibiotic resistance gene and one inactive gene will be able to growunder selective conditions. Under these same conditions, one can easilydistinguish inactive marker from mixed active/inactive. It will beunderstood by one of skill in the art that this type of a qualitativeanalysis is merely a convenience, and not essential to the practice ofthe invention. Methods of quantitative analysis, e.g. ELISA, RIA, etc.,that can distinguish between the amount of marker produced by one activegene and the amount of marker produced by two active genes (or multiplesthereof) may also be used. Such quantitative methods permit either thedetection of cells having only active marker from cells having a mixtureof active and inactive, or the detection of cells having only inactivemarker from cells having a mixture of active and inactive.

The I vector, which is methylated on the adenine of the GATC recognitionsite, can be replicated in most common laboratory strains of E. coli.Other bacterial hosts that modify DNA at this site may also be used forpreparing the I vector DNA. Generally, DNA replicated in non-bacterialcells will require an additional ex vivo methylation step, usingpurified DNA methylases. Substantially all of the GATC sites in the Ivector will be methylated.

The A vector must be replicated in a host that lacks this DNAmodification system. Suitable E. coli dam- strains include JM110,described in Janisch-Perron (1985) Gene 33:103-119. A vectors replicatedin non-bacterial host cells, e.g. yeast, mammalian cell culture, etc.may also be used.

Convenient vectors for preparation of single stranded DNA arederivatives of M13 phage, see Messing (1983) Meth. in Enzym. 101:20. M13is a filamentous bacteriophage, and is commonly used in researchlaboratories. Derivatives of the wild-type phage are known in the art,and commercially available from a number of sources. M13 phage (+)strand DNA can be isolated from phage particles. Double stranded phageDNA is isolated from infected cells, and the (−) strand can be isolatedfrom the double stranded form by various strand separation methods knownin the art, e.g. columns, gels. Alternatively, the (+) strand may beused in combination with the double stranded form. E. coli strainssuitable for M13 replication include JM101, JM105, JM107, JM109, etc.Vectors carrying the M13 origin of replication (phagemids) and capableof producing single stranded or double stranded DNA are known in the artand widely available.

The strands of the A and I vector that participate in forming the testvector are substantially complementary. To form the test vector, the Aand I vectors are linearized, denatured if necessary, and annealed toeach other. Various methods are known for linearizing molecules, e.g.digestion with restriction enzymes, etc. Methods of denaturing andannealing DNA are well known in the art, and need not be described indetail. The two termini may have blunt ends, or complementaryoverhanging ends. The annealed, heteroduplex DNA is circularized by aligation reaction, using any suitable ligase, e.g. T4, E. coli, etc.,using conventional buffers and conditions. Generally, the quantity ofheteroduplex DNA formed will be sufficient to detect in a standardtransformation reaction, e.g. at least about 0.1 picograms of DNA.

Where double stranded vectors are used, the vectors must be linearizedand denatured prior to the annealing step. In addition, it is desirableto remove the homoduplex A and I vectors after annealing and prior totransformation, in order to avoid a high background of transformants.One convenient method of performing this step takes advantage of thedifferential methylation of the two vectors. Restriction enzymes areknown in the art that will cleave homoduplex unmethylated DNA, e.g. MboI, and homoduplex methylated DNA, e.g. Dpn I, but will not cleaveheteroduplex DNA having one methylated and one unmethylated strand. Thedouble stranded A and I vectors are denatured, combined, and reannealed,leaving a mixture of homoduplex DNA (A vector, I vector) andheteroduplex DNA (A/I vector). The mixture is then treated with themethyl specific restriction enzymes. The homoduplex DNA is cleaved, andthe heteroduplex is not. The heteroduplex DNA is then used in subsequentsteps of the method.

The Test Sequence

The test sequence is a heteroduplex of X and Y, as previously described.X and Y are substantially complementary, and anneal with each other.Generally, the sources of the X and Y strands will be closely related,e.g. individuals of a single species, individuals of closely relatedspecies, germline and somatic tissue from a single individual, inbredstrains of a species, etc. The test sequence may be derived from anysource, e.g. prokaryotic or eukaryotic, plant, mammal, insect, etc. Thesubject method is particularly useful for the analysis of complexgenomes, such as those found in higher plants and animals. The test DNAsequence will usually be of at least about 20 nt in length, and usuallynot more than about 10⁴ nt in length. The upper limit on length isdetermined by the ability of the MMR host to co-repair the strand.

In order to initiate co-repair of the marker gene, there must be atleast one “initiating mismatch” in the test sequence. An initiatingmismatch is a deletion, insertion or substitution of from one to fourcontiguous nucleotides. A loop of five or more contiguous nucleotideswill not initiate repair. Multiple non-contiguous mismatches may bepresent in the test sequence. Generally, the test sequence will have atleast about 90% identity between the two strands. Initiation ofco-repair will proceed as long as one initiating mismatch is present.

Various methods may be used to generate the X and Y strands. Isolatingand amplifying DNA sequences are known in the art. X and Y may be cDNAfrom a reverse transcriptase reaction, a restriction fragment from agenome, plasmid, YAC, virus, etc.; an amplification product frompolymerase chain reaction (PCR), etc. An important limitation to the useof PCR products is the choice of thermostable polymerase. Polymeraseshaving a 3′ to 5′ exonuclease activity, e.g. proofreading function, arepreferred. Useful thermostable polymerases with proofreading capabilitythat are known in the art include those isolated from Thermococcuslitoralis, Pyrococcus furiosis, and Thermus thermophilus. Commerciallyavailable Thermus aquaticus polymerase has been found to introduce asignificant number of errors into the amplified DNA, and will generallybe unsuitable for all but very short, e.g. less than about 500 nt.,sequences.

Where the test sequence is obtained from an in vitro amplificationreaction, it may be desirable to methylate the amplification product,using conventional enzymes and methodologies.

A number of techniques are known in the art for isolating singlestrands, or for denaturing double stranded DNA. For example, a reversetranscriptase product may be treated with ribonuclease to leave only theDNA strand. Strand separation gels are known in the art and may be usedto separate the two strands of a DNA molecule. PCR may be performed withone primer conjugated to a molecule with a binding partner, such asbiotin, haptens, etc. The PCR reaction is then denatured, and bound to asolid substrate conjugated to the binding partner, e.g. avidin, specificantibody for the hapten, etc. The test DNA may be replicated as a singlestranded entity, e.g. M13 phage, phagemid, etc. The X and/or Y sequencemay be restriction fragments, PCR products, or other double stranded DNAmolecules, that are denatured according to conventional methods.International application PCT/US93/10722 describes one method forgenerating heteroduplex DNA suitable for mismatch testing.

There are several different methods that may be used to attach the testsequence DNA to the vector(s). In one method, the double stranded A/Ivector is ligated to double stranded X/Y test sequence DNA. This methodligates double stranded heteroduplex A/I vector to double strandedheteroduplex X/Y test DNA. The two double stranded DNA molecules arecombined. It is convenient to have a short, complementary overhang onthe termini of the X/Y, and the A/I molecules, such as those formed bydigestion with various restriction endonucleases or by the ligation ofspecific linkers to the termini, where the vector and the test sequencewill anneal to each other. Preferably, a different overhang will bepresent on each termini of one molecule, so as to preventself-circularization of the vector. Blunt ends may also be used, inwhich case it may be desirable to phosphatase treat the vector ends toreduce self-circularization. The molecules are ligated to form acircular dsDNA, which is then used in subsequent steps.

In another method, X and Y DNA is ligated into the A and I vectors in aseparate cloning step, and the chimeric DNA strands are used to form theA/I heteroduplex molecule. The X and Y sequences may be separatelycloned into the A and I vectors, using conventional recombinant DNAmethods (see Sambrook et al., supra.). Either strand may go into eithervector. The chimeric molecules may then be replicated as previouslydescribed, to provide methylated and unmethylated strands. The chimericmolecules are linearized, denatured if necessary, annealed, and ligatedas described above to form the A/I vector.

In a preferred method, test DNA from only one source (X) is cloned intothe A or I vector, to form a chimeric molecule. While either the Ivector or the A vector may be such a chimera, conveniently, the A vectorwill contain a copy of the test sequence. Such a vector may be referredto as a “standard” vector. A single standard may be used in a reaction,or multiplex reactions may be performed, where a plurality of standards,each comprising a distinct test sequence, are hybridized in a singlereaction. The multiplex reaction may combine two or more standards,usually at least about 10 standards, more usually at least about 100standards, and may combine as many as 10,000 or 100,000 standards.

The single stranded standard vector may be combined in a hybridizationreaction with the I vector and the Y test sequence, to form aheteroduplex, where the strand are then annealed and ligated.

In such cases, it will be desirable to clone only one strand of the testsequence into a vector, and have the other strand of the test sequencebe provided separately. Using conventional recombinant DNA techniques,the test sequence (arbitrarily designated X) is cloned into the A or Ivector. Either vector may be recipient of the X DNA. For some uses ofthe method, it may be advantageous to use the A vector as recipient,because the final DNA product, after transformation and methyl mismatchrepair, will then be corrected to have the sequence of the Y(methylated) strand, thereby allowing isolation and further growth ofthe Y DNA. If the vector will be grown as a single stranded entity, thenthe complementarity of the strands must be selected so that X and Y willbe capable of hybridizing.

The chimeric A or I vector, containing X DNA, is linearized and annealedto the complementary vector, to form a heteroduplex A/I vector having asingle stranded X region. Y DNA is combined with the heteroduplexvector, and annealed to X. It will be understood by one of skill in theart that a single annealing reaction may be performed with these threemolecules. Y may be denatured double stranded DNA, e.g. a PCR product,fragment of genomic DNA, etc., or may be single stranded, e.g. cDNA,etc. The three strands (I, AX and Y) are then ligated.

Transformation and Detection

The test vector, heteroduplex A/I vector ligated to X/Y test sequenceDNA, is transformed into a suitable bacterial host. Most bacterialspecies have an active methyl mismatch repair system, and can thereforebe used as an MMR host. Suitable species include E. coli and other gramnegative rods, such as Pseudomonas, Erwinia, Shigella, Salmonella,Proteus, Klebsiella, Enterobacter and Yersinia. Other species ofinterest include B. subtilis, Streptomyces, etc. The genetics and growthrequirements of E. coli are well known, and in most cases it will be thepreferred host. Transformation techniques are well known, for examplesee Hanahan (1985) in: DNA Cloning, Vol. 1, ed. D. Glover, IRL PressLtd., 109.

The transformed bacteria are generally grown under selective conditions,where only those cells able to express a vector encoded selective markercan proliferate. Preferably the test vector will include a selectivemarker, such as antibiotic resistance, for this purpose. Thetransformants may be grown in a suitable culture medium, e.g. LB broth,SOB broth, 2YT, etc., as a liquid culture, on plates, etc. In somecases, the growth medium will also include any substrates required forshowing of the detectable marker.

The determination of transformants expressing active and inactive markeris then made. The method of determination will vary with the specificmarker used, as previously discussed. In one embodiment, plates oftransformants are counted for colonies having a positive or negativecolor change, such as cleavage of indolyl-β-D-galactoside to produce ablue color, or expression of luciferase. In another embodiment, replicaplates are made, and it is determined whether cells from individualcolonies are capable of growing in a selective medium. Transformantsgrown in liquid culture may by stained, for example with antibodiesspecific for the selectable marker, and analyzed by flow cytometry todetermine the number of cells expressing active marker.

Transformants that lack active marker had an initiating mismatch in thetest sequence. An increase in the percentage of transformants that lackactive marker, compared to a control, perfectly matched test sequence,is indicative of a mismatch. The transformed bacteria that lack activemarker are growing the “corrected” test vector, where both strands ofvector DNA will have the sequence of the originally methylated strand.The transformed bacteria that express active marker will generally havea mixture of A and I vector. Vector DNA may be prepared from thetransformants, and used for further purification and characterization.

Applications of the Method

The subject method is useful for analysis of DNA polymorphisms, mutationand for isolation of variant sequences. A number of applications for thesubject method are based on detection of sequence polymorphisms in asingle, known DNA sequence. For example, in prenatal diagnosis one mightwish to determine whether a mutation in a particular gene, e.g.hemoglobin, dystrophin, etc., is found in a fetal DNA sample. Many tumorcells contain a mutation in one or more oncogenes and/or tumorsuppressor genes. Determining whether a particular gene is altered in atumor cell sample is therefore of interest. Determining the occurrenceand frequency of sequence polymorphisms in a population is important inunderstanding the dynamics of genetic variation and linkagedisequilibrium.

To perform this type of analysis, a control (X) copy of the sequence ofinterest is cloned into the A or I vector, usually A vector. Where agene is known to be polymorphic, several different vectors, each havinga different allelic form, may be used. The Y sequence is obtained from asuitable source of DNA, depending on the type of analysis beingperformed. The Y sequence may also be cloned into a vector. In apreferred embodiment, however, a heteroduplex is formed of AX and Istrands combined with single stranded Y DNA, where Y may be a denaturedPCR product, cDNA etc. X and Y are annealed, and a ligation is performedto produce the test vector.

For genetic testing, one may set up a panel of A or I vectors havingdefined regions of a chromosome, for example the BRCA1 gene, or CF gene,where a copy of the gene sequence is cloned into the vector. Similarly,for identification of variation involved in clinical phenotypes, one mayset up a panel of A or I vectors carrying many fragments to test forSNPs, or gene variations. Due to allelic variation, it may be necessaryto compare several sets of control vectors. The length of some genes maynecessitate a series of vectors, in order to cover the entire region.The Y sequence DNA is obtained from the individual being tested, usingany convenient source of DNA. The Y sequence may be added to the AX/Ihybridization reaction, or may be cloned into the I vector in a separatereaction. Hybridization of the panel of X sequence vectors with thecorresponding Y sequences may be performed in parallel, or in amultiplex reaction. The presence of specific sequences is thencorrelated with the presence or absence of active marker gene. One canthen determine, for large regions of DNA, or a large number of geneswhere an individual sequence varies from a standard, control sequence.

The resulting colonies from the above procedure will be a mixture ofactive marker expressing, having a DNA sequence identical to the controlsequence, and lacking active marker, where there was an initiatingmismatch in the test sequence. In order to analyze the results, it maybe desirable to determine the frequency of these two populations. Thismay be accomplished by separating the active and inactive colonies intotwo different pools. Separation may be accomplished by picking colonies,flow cytometry, column separation based on binding of the marker,immunomagnetic bead separation, etc. Vector DNA isolated from thesepools is digested with an appropriate restriction endonuclease torelease the insert. Gel electrophoresis may then be used to quantitatethe amount of insert DNA in each pool, using the vector band as aninternal standard, from which the proportion of variant and identicalclones can be determined. Acrylamide gels (or other separation methods)can be employed. Alternatively, the insert DNA from each of the poolsused as a hybridization probe on a hybridization filter or microarray offragments corresponding to the fragments being tested. The ratio ofsignal intensity from hybridization with the active and inactive pool ofinserts can be used to determine the proportion of variant and identicalsequences. This allows the simultaneous analysis of sequence variationfor many different fragments.

The nature of the X/Y sequences varies. In one embodiment the testsequences will include all the coding regions and their regulatoryelements for a particular organism, e.g. human, yeast, etc. In anotherembodiment they are polymorphic markers that can be used for geneticmapping. In yet another embodiment they are one or several genes thatare tested in a clinical setting to for the purposes of improving thediagnosis, prognosis, or treatment for a patient.

This multiplexing can be augmented by assessing the genotype of multipleindividuals at the same time, for a particular fragment or geneticsequence of interest. Alternatively, multiple samples may be taken froman individual to determine the extent of somatic mutation in a cellpopulation, e.g. tumor cells, etc. The sample nucleic acid may be anamplification product, cloned fragment, etc. By assessing the geneticvariation in a population one can estimate the frequency of variation ina particular population in a variety of genes in one experiment. One canidentify genes related to clinically relevant phenotypes by identifyingthose genes that have a higher frequency of variation in the populationof interest as compared with the normal population. In addition thisapproach can be used to identify fragments carrying variations andtherefore can be useful as for SNP testing.

In other applications of the method, one may wish to isolate variants ofsequences, particularly genomic sequences. In some cases, the controlsequence will be only partially characterized. For example, many geneticdiseases or conditions are known only by their phenotype and general mapposition, e.g. a high predisposition to breast cancer, obesity, etc.Localization of the gene to a particular map region, or a YAC clone,still leaves hundreds of thousands of bases of DNA containing thepotential gene candidate. MRD provides a means of identifying andisolating the variant sequence.

DNA is isolated from two sources. The DNA may be from a YAC or BACinsert, a restriction fragment from a human chromosome, etc. One sourceof DNA will have the putative variant sequence, and the other will havethe control sequence, e.g. wild-type. Preferably the two sources will berelated, e.g. inbred mouse strain, tissue samples from an individual,human parent or sibling, etc. The transformed cells are useful as asource of cloned DNA.

In one method, the two DNA samples are cloned into the I and A vectors,respectively, to provide inserts of not more than about 10⁴ nt inlength, and usually at least about 10² nt in length. The vectors areseparately replicated in methylation positive and methylation negativeconditions, either as single or double strands. The two vectors are thenlinearized, denatured if necessary, annealed, ligated, and transformedinto an MMR host, as previously described. There will be a large numberof transformants that represent perfect matches, and will express activemarker gene. The transformants that lack an active marker have amismatch between the two DNA sources, and are candidates for clones ofthe variant sequence.

The ability of MRD to isolate DNA having a variant sequence can be usedin “multiplexing” procedures, where multiple DNA fragments are analyzedin a single reaction. Multiplex reactions may be set up for specificfragments of DNA or regions of a chromosome, etc. In multiplexreactions, generally two cycles of MRD will be performed. The firstround of MDR provides a number of bacterial colonies having variant oridentical allele(s) from a pool of DNA fragments. The second round ofMDR further enriches for the variant sequences.

Regions of DNA may be compared in multiplex reactions. One or manydifferent fragments may be isolated in a single reaction. Generally DNAfrom one source will be fragmented by a suitable method, e.g.restriction endonuclease digestion, etc., cloned into the appropriatevector, hybridized with the other vector as well as DNA from the othersource, and a first round of MRD analysis performed in a singlereaction. Colonies having inactive marker after the first round areenriched for variant sequences. DNA isolated from these colonies may becompared to the control sequence, using additional round(s) of MRD tofurther enrich for variants. The majority of inactive colonies from thesecond round will carry DNA sequences that differ from the control.Where error prone polymerase was used to generate DNA, the method of“cleaning” described below may be used to enrich for true variants.

An alternative approach to isolating variant sequences is as follows.Two DNA samples, e.g. YAC, plasmid, restriction fragment, etc.,containing the region of interest are cleaved with a restrictionendonuclease into fragments of not more than about 10⁴ nt. The twosamples are combined, denatured, and allowed to anneal. The X/Y mixtureis then annealed and ligated into a heteroduplex A/I vector havingcompatible ends. The mixture is transformed into an MMR host. Anytransformants lacking active marker will represent a mismatch betweenthe two DNA sources.

Isolation of variant fragments can be done for many fragments from manypeople in the same experiment. For example PCR from a pool ofindividuals can be performed for many fragments. These PCR products canbe annealed and ligated into a heteroduplex A/I vector. Alternativelythey can be annealed to an A vector with an X sequence already ligatedto it. Two MRD procedures might be performed as described above toenrich for the variant fragments. This approach can be useful inidentifying in a population the fragments carrying variations andtherefore that can be used as genetic markers. In addition this approachmay identify variations in coding regions that may be involved inspecific clinical phenotypes. This approach can be performed withdifferent populations (one experiment per population) in order toisolate those variations that are specific to a specific population. Inother words MRD can be used to identify rare alleles in a population fora large number of genes. An analogous application is the identificationof rare alleles produced by somatic mutations or sperms in oneindividual. Examples of this include identification of rare alleles in afraction of tumor cells, precancerous changes in a pool of normal cells,mutations caused by environmental mutagens, or somatic mutations thatmay be relevant in processes such as immune diseases or aging.

In addition to the use of MRD for identification of human geneticvariation involved in clinical phenotypes, e.g., phenotypes affectingthe development, progression, or treatment of disease, MRD can clearlybe used to test variation in nonhuman species. Identification ofvariations leading to phenotypes in mice, drosophila, yeast and otherspecies is of concern to researchers. In addition, identifyingvariations in human pathogen like HIV virus of Mycobacteriumtuberculosis can have important clinical consequences. Finally otheruses of MRD can be in identifying variation relevant to farmingphenotypes, e.g. variations leading to increased milk production in cowsor prolonged freshness in tomatoes.

MRD may be used in conjunction with Taq polymerase to enrich formolecules that are free of PCR-induced errors. Following this “cleaning”protocol, the cloned PCR products is isolated for further analysis. Theproducts of a Taq PCR reaction are cloned into the control and testvectors, and are then hybridized and transformed. The majority oftransformants containing Taq PCR-induced errors will present asheteroduplex molecules containing a mismatch and will not produce activemarker. In contrast, those PCR products with no PCR-induced errors willcontain no mismatches and will produce active marker. These colonies canbe isolated, and if desired, undergo a second round of cleansing. Asimilar protocol may be used to isolate non-variant sequences from apopulation.

It is contemplated that a kit will be provided for the practice of thesubject invention. At a minimum, the kit will contain A and I vectors.The vectors may be single or double stranded. Single stranded vectorsmay be pre-annealed in an A/I heteroduplex. Competent host bacteria forgrowing unmethylated and methylated vector may also be included, as wellas an MMR host strain. For analysis of specific DNA sequences, e.g.oncogenes, tumor suppressor genes, human β-hemoglobin, cDNA and genomiccopies of BRCA1 and BRCA2, a panel covering the human dystrophin gene,etc., a kit may be provided where a chimeric A vector is provided,containing the X (control) sequences. The A and I vector in this casemay also be pre-annealed, to form an AX/I heteroduplex. Such a kit mayalso include specific primers for amplifying the Y sequence DNA, andoptionally, thermostable polymerase.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the cell” includes reference to one or more cells andequivalents thereof known to those skilled in the art, and so forth. Alltechnical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acomplex” includes a plurality of such complexes and reference to “theformulation” includes reference to one or more formulations andequivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the methodsand methodologies that are described in the publications which might beused in connection with the presently described invention. Thepublications discussed above and throughout the text are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior invention.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight, andpressure is at or near atmospheric.

Experimental EXAMPLE 1

Two pUC-derived plasmids, the A plasmid (pMF200) and the I plasmid(pMF100), are employed in the MRD procedure. A map of the plasmids isshown in FIG. 3. These plasmids are identical except for a five bpinsertion into the Lac Zα gene of pMF10O. This insertion results inwhite colonies when bacteria transformed with the I plasmid are grown onLB plates supplemented with indolyl-β-D-galactoside (Xgal) andisopropyl-β-D-thiogalactoside (IPTG). In contrast, bacteria transformedwith the A plasmid result in blue colonies when grown under theseconditions.

The initial step of the MRD procedure consists of cloning one of two DNAfragments to be screened for differences into the A plasmid and cloningof the second DNA fragment into the I plasmid. The A plasmid constructis then transformed into a dam- bacterial strain, resulting in acompletely unmethylated plasmid while the I plasmid construct istransformed into a dam⁺ bacterial strain, resulting in a fullymethylated plasmid. The two plasmids are then linearized, denatured, andreannealed, resulting in two heteroduplex and two homoduplex plasmids.Following digestion with Mbo I and Dpn I, which digest onlyhomoduplexes, the remaining hemimethylated heteroduplexes arecircularized, transformed into E. coli, and plated onto agarsupplemented with Xgal and IPTG.

In the absence of a mismatch between the two test DNA fragments, thefive nucleotide loop in the Lac Zα gene, resulting from heteroduplexformation between the I and the A plasmids, is not repaired by themismatch repair system. Subsequent plasmid replication produces both Iand A plasmids in a single colony, leading to a blue color. In contrast,if a mismatch is present in the heteroduplex DNA, a co-repair eventtakes place that involves both the mismatch in the DNA as well as thefive nucleotide loop in the Lac Zα gene. In this case, the unmethylatedLac Zα gene on the A plasmid is degraded, and replaced by the Lac Zαgene from the methylated strand of the I plasmid, resulting in a whitecolony. The data show that co-repair of a mismatch and the Lac Zα genein the MRD system occurs even when the distance between them is greaterthan 5 kb.

Methods

The MRD vectors. pMF100 and pMF200 are derived from pUC19, with themultiple cloning site displaced from the Lac Zα region. In addition, theMRD vectors contain the Bgl I fragment (2166-472) and most of themultiple cloning site of pBluescript (Stratagene, La Jolla, Calif.). Thecloning sites of the MRD vector do not have sites for the restrictionendonucleases Xbal, Spel, BamHI, Smal and Apal. The EcoRI site is notunique. pUC19 multiple cloning sites, nucleotides 400-454, were replacedusing 70 nucleotide long oligonucleotides with a sequence containingfour GATC sites. In addition, the site replacing the pUC19 multiplecloning sites in pMF200 has a 5 bp insertion as compared to pMF100,creating a non-functional Lac Zα in pMF200. The label “loop” in FIG. 3indicates this difference.

Formation of heteroduplex DNA. DNA from the unmethylated and methylatedplasmids are linearized, denatured, and reannealled. The resultingmolecules are fully unmethylated A plasmid homoduplexes, fullymethylated I plasmid homoduplexes, and hemimethylated heteroduplexes.The mixture is digested with Mbol, which digests fully unmethylated DNA,and DpnI, which digests fully methylated DNA. Only the heteroduplex,hemimethylated DNA is left.

EXAMPLE 2

As an initial test of the sensitivity and specificity of the MRD system,a single nucleotide mismatch was detected in a 550 base pair DNAfragment derived from the promoter of the mouse beta globin gene (Myerset al. (1985) Science 229:242). MRD was used to compare this DNAfragment, which contains a T at position −49 (relative to the functionaltranscription start site of the gene) with a second DNA fragmentidentical in sequence except for at C position −49. The mismatch waslocated about 700 base pairs from the five nucleotide Lac Zα loop in thevector. Comparison of the two DNA molecules by using MRD resulted in 90%white colonies. In contrast, comparison of the same two DNA moleculeswith no mismatch (−49T/−49T), resulted in only 7% white colonies. Thedata is shown in Table 1.

TABLE 1 Detection of Known Point Mutations using MRD Sequence Distancefrom % White (Inactive) Variation* Fragment Size{circumflex over ( )}Loop{circumflex over ( )} Colonies^(@) None¹ 0.55 N/A  7 G_C¹ 0.55 0.789 A_T¹ 0.55 0.7 84 G_T¹ 0.55 0.7 82 A_C¹ 0.55 0.7 82 C_T¹ 0.55 0.7 90None² 2.0 N/A  8 A_C² 2.0 0.4 35 None³ 2.2 N/A 10 C_T³ 2.2 2.3 83 G_A³2.2 2.1 86 C_T³ 2.2 1.6 81 T_C³ 2.2 1.8 80 *A_T variation means that atthe only position of variation between the two fragments compared, thedamgrown variant has an A and the dam+ grown variant has a T at the sameposition on the same strand. Therefore, mismatches produced in such anexperiment are A/A and T/T. {circumflex over ( )} in kilobases. ^(@)Atleast 250 colonies were counted to determine the percentage. ¹Experimentusing a fragment of the mouse beta globin gene. ²Experiment using afragment of the human agouti gene. ³Experiment using fragment of humancystathionine beta synthase gene, at positions 341, 502, 992, and 833,respectively.

Comparison of all possible single nucleotides mismatches at position −49using MRD revealed proportions of white colonies ranging from 80% to90%. These results demonstrate that MRD can detect all of the differentDNA variations possible at this position with high efficiency.

The MRD system was used to detect a total of five additional singlenucleotide mismatches in two different DNA fragments, shown in Table 1.Four of these mismatches are at different nucleotide positions in thehuman cystathionine beta synthase gene (Kruger and Cox (1995) HumanMolecular Genetics 4:1155). The remaining one mismatch represent singlenucleotide changes in the human agouti gene (Wilson et al. (1995) HumanMolecular Genetics 4:223). In each case, a single nucleotide mismatchwas detected.

A mismatch was detected even when it was as far as 2.3 kb from the LacZα loop. Since the proportion of white colonies was greater than 50%,co-repair of the mismatch and the loop on the unmethylated strandoccurred irrespective of which side of the mismatch was relative to theloop.

To determine whether the efficiency of mismatch detection would remainhigh if the distance between a mismatch and the vector loop was evenlarger, the following experiment was performed. A 9 kb test DNA fragmentderived from lambda bacteriophage was cloned into the MRD plasmid systemand compared with the same test DNA containing a two base pair insertionlocated 5 kb from one end of the fragment. Addition of the two base pairmismatch resulted in 70% white colonies, as compared to 10% whitecolonies in the absence of the mismatch. These results indicate that MRDcan detect a mismatch in 10 kb of DNA.

EXAMPLE 3

MRD was used to detect unknown mutations in genomic DNA fragmentsgenerated by the polymerase chain reaction (PCR). PCR is a practicalmethod for obtaining a particular genomic DNA fragment of interest frommany different individuals. Recent advances in PCR technology makes itpossible to isolate DNA products greater than 10 kb in length (Barnes(1994) P.N.A.S. 91:2216; Cheng et al. (1994) P.N.A.S. 91:5695). However,the introduction of errors during the PCR reaction severely limits theuse of individual cloned PCR products. In an effort to overcome thislimitation, an MRD protocol was developed to enrich for molecules thatare free of PCR-induced errors. Following this “cleaning” protocol, thecloned PCR products can be compared for DNA sequence differences byusing the MRD procedure described above.

The basic principle underlying the MRD cleaning protocol is the factthat any single PCR-induced mutation will make up a very small fractionof all the molecules generated by PCR. As a result, when the products ofa PCR reaction are cloned into the A “blue” and the I “white” MRDvectors and assayed as described above, the majority of productscontaining PCR-induced errors will present as heteroduplex moleculescontaining a mismatch and will produce white colonies. In contrast,those PCR products with no PCR-induced errors will contain no mismatchesand will result in blue colonies. Given that not all mismatches arerepaired with 100% efficiency, some blue colonies can be expected tocontain PCR-induced errors following the first round of enrichment.However, if blue colonies are isolated and used in a second round of MRDcleaning, those molecules containing PCR-induced errors can be reducedeven further. Since each blue colony contains both a blue MRD plasmidand a white MRD plasmid, the second round of MRD cleaning is carried outas follows. Plasmid DNA isolated from blue colonies following the firsround of cleaning is used to transform both dam− and a dam+ bacterialstrains. Although both blue and white colonies resulted from eachtransformation, only the blue colonies are isolated from the dam-transformation, and only the white colonies are isolated from the dam+transformation. Plasmid DNA is prepared from such colonies andheteroduplexes are isolated as described above. Blue colonies arisingfrom transformation with these heteroduplexes are further enriched forthe products free of PCR-induced error. In an experiment in which 75% ofmolecules contain one or more PCR-induced errors following PCR, assuming95% efficiency of mismatch repair and 10% frequency of white colonies inthe absence of a mismatch, the expectation would be 10% blue coloniesfollowing one round of MRD enrichment, with 66% of the molecules in suchcolonies free of PCR-induced errors. If the plasmid DNA from the bluecolonies were used for a second round of MRD enrichment, the expectationwould be 41% blue colonies, with 96% of the molecule in such coloniesfree of PCR-induced errors.

As a test of the practicality as well as the efficiency of the MRDcleaning protocol, a 2 kb human chromosome 21-specific PCR product wasisolated from each of the two chromosome 21 homologues of a singleindividual. The two chromosome 21 homologues were separated from eachother in independent hamster-human somatic cell hybrid clones. GenomicDNA isolated from these somatic cell hybrid clones was the source of PCRproducts. When the PCR products derived from each homologue werecompared using MRD as described above, approximately 10% blue colonieswere observed in each case.

Following two rounds of MRD cleaning, the proportion of blue colonies as60-80%, data shown in Table 2. In contrast, when these “cleaned” PCRproducts derived from the two homologues were compared with each otherby using MRD, approximately 90% of the resulting colonies were white,indicating the presence of at least one single base difference in the 2kb PCR products derived from the two different chromosome 21 homologues.The DNA sequence variation in the PCR products was independentlyverified by restriction enzyme digestion. These results demonstrate thatMRD can be used to enrich for PCR products that are largely free ofPCR-induced errors, and that such products can be used in conjunctionwith MRD to detect human DNA sequence variation.

TABLE 2 Percentage of Inactive Colonies in Different Comparison withPlasmids containing 2 kb PCR Products from two Somatic Cell HybridsVariants Compared* Percentage of Inactive Colonies^(#) 1/2 >90 2/2 >90A1/A1 70 A2/A2 64 AA1/AA1 38 AA2/AA2 21 AA1/AA2 >90 AA2/AA1 >90 *1 and 2represent products from the two hybrids. 1/1 represents comparison of Avector grown in a dam− strain and containing the PCR product from hybrid1 to I vector grown in a dam+ strain and containing the PCR product fromhybrid 1. A1/A1 represents the comparison of A vector grown in dam−host, obtained from the active colonies of comparison 1/1, to I dam+grown vectors obtained from the same source. AA1/AA1 represents thecomparison of A dam− grown # vectors obtained from the active coloniesof the comparison A1/A1 to I dam+ grown vectors from the same source.Finally, AA1/AA2 represents the comparison of A dam− grown plasmidsobtained from the active colonies of the comparison A1/A1 to I dam+grown vectors obtained from the active colonies of the comparison A2/A2.

It is evident from the above results that the subject invention providesfor an efficient, simple method of detecting mismatches between two DNAsequences. The method provides a means of simply detecting the presenceof a mismatch, or can be used to isolate copies both matched andmismatched DNA. MRD is useful to determining somatic changes in genesequence, identifying germline mutations for prenatal or other geneticscreening, for human gene mapping, and for cloning mutations. A majoradvantage of MRD is the potential of this system to analyze manyfragments simultaneously in a single experiment, allowing the detectionof mutations in a region representing hundreds of kilobases of DNA, orfor genotyping many loci simultaneously. MRD provides a powerfultechnique for the detection of unknown mutations, the detection of DNAvariation in large genomic regions, and high-throughput genotyping.

EXAMPLE 4 Use of Cre-Lox as a Detectable Marker

Construction of the Standards:

MRD utilizes two vectors that are identical except for a five base pairdeletion in the gene coding for Cre recombinase on one of the vectors.DNA fragments are cloned in the vector containing the wild type Cre.These clones, referred to as standards, are made only once and serve assequence comparison templates for sequences from each person that is tobe tested. Standards are grown in an E. coli host that is deficient inmethylation, and subsequently unmethylated single stranded DNA isobtained.

Heteroduplex Preparation for DNA Variation Screening:

In order to perform the screening for DNA variations, DNA fragments thatare to be tested are amplified from each individual. After in vitromethylation of the PCR products, single stranded DNA from all of thestandards are pooled and added to the tube containing the PCR products.Linearized vector containing the 5 base pair deletion in the Cre gene isalso added to the same tube. The three components (the PCR products, thesingle stranded standards, and the linearized Cre deficient vector) aredenatured by NaOH and reannealed by neutralization. This process createsheteroduplexes between the unmethylated single stranded standard, itscomplementary PCR product and the linearized Cre deficient vector, shownin FIG. 6.

Mung Bean nuclease is added to degrade remaining single strandedcomponents. Taq ligase is then added to create closed-circlehemimethylated heteroduplexes. At this point a single tube contains allof the heteroduplexes corresponding to the standards and the genefragments that are being tested. In a single reaction mixture theheteroduplex DNA is transformed into an electrocompetent E. coli strain(Mutation Sorter, MS) engineered to carry on its chromosome a cassetteof a tetracycline resistant (tetR) and streptomycin sensitive (strepS)genes flanked by two lox sites.

Separation of Variant and Non-Variant DNA Fragments:

Those heteroduplex molecules carrying no mismatch (i.e., no variationbetween the standard and the DNA fragment that is being tested)replicate normally, and plasmids carrying both the active and inactiveCre will be present. The active Cre recombines the cassette between thetwo lox sites leading to the loss of the tetR and the strepS genes. Thisrenders the cell tetracycline sensitive and streptomycin resistant, andhence it will grow in the presence of streptomycin but not tetracycline.

The presence of a mismatch (i.e., if there is a variation between astandard and the DNA fragment that is tested) in the heteroduplexmolecules leads to the repair of such mismatches. In the process ofrepairing the mismatch the unmethylated strand carrying the active Cregene is degraded and the strand carrying the inactive Cre is used as atemplate to be copied. As the result, the cell transformed with amismatch heteroduplex is devoid of any Cre activity, permitting the cellto retain its tetR and straps cassette and therefore grow in presence oftetracycline and not streptomycin.

By growing the transformation mixture in two tubes containing eithertetracycline or streptomycin, fragments containing a variation and thosethat do not contain a variation are isolated, respectively. Theevaluation of DNA variation detection is reduced to identifying whichfragments are present in which pools. This task may be done in multipleways including gel electrophoresis and hybridization.

IDENTIFICATION OF VARIANT AND NON-VARIANT DNA FRAGMENTS

Using ABI sequencing gels: DNA from each pool is digested withrestriction enzymes to release the fragments being tested. Samples ofthe two restriction digests are fluorescently labeled and run on an ABIsequencing gel. The presence or absence of variation in a fragment isassessed by determining the pool where the specific fragment is present.This can be achieved because the different fragments are separated fromeach other according to their size.

Using DNA microarray technology: In this procedure all of the fragmentsthat are represented in the standards are dotted onto slides.Subsequently the DNA obtained from the tetracycline containing cultureand streptomycin containing culture are fluorescently labeled and usedas hybridization probes. The probe from the tetracycline culturehybridizes to the spots corresponding to the DNA fragments that containvariation; the probe from the streptomycin culture hybridizes to thespots corresponding to DNA fragments that contain no variations. Othermethods for the analysis of the fragment content of each pool can beutilized, including mass spectroscopy.

In order to achieve the goals of identifying variations involved inclinically relevant phenotypes, many genes need to be tested. Thistremendous task is greatly facilitated by MRD's ability to multiplex.Many researchers have used whole cDNA content of the cell as the probeonto a microarray containing more than 10,000 targets. It is thereforebe a relatively simple task to use a pool of 4,000 fragments as a probeon a microarray containing 4,000 targets. This will allow for thetesting of 4,000 fragments simultaneously, although more samples couldbe multiplexed. In order to test the entire coding regions of the genesof one human individual, one could test 400,000 fragments with anaverage size of 300 bp each. It will then take 100 MRD reactions toachieve that task.

Disease-causing variations, by definition, have increased frequency inthe patient population than in controls. Since construction ofhaplotypes and knowledge of every individual's genotype are notnecessary to identify the disease-causing variations, patients orcontrols can be pooled and tested to estimate the frequency of differentDNA variations in each population. MRD has the potential tosimultaneously estimate the frequency of many variations in apopulation. First, genomic DNA from many individuals is physicallypooled and used as template for subsequent MRD steps. The frequency ofevery variant fragment is estimated by determining its prevalence in thetwo pools obtained at the end of the procedure. Obtaining frequencies ofdifferent variant gene fragments in different populations, e.g.,patients and controls, can quickly identify the fragments carryingdisease-causing variations.

In order to demonstrate MRD's potential to multiplex, we have appliedthe MRD procedure to the identification of DNA sequence variation in 13DNA fragments randomly selected from a group of published polymorphicSequence Tagged Sites (STSs). In brief, standards were made for eachSTS, and heteroduplexes were made between a mixture of the standards andDNA fragments amplified from each individual tested. Heteroduplexes weretransformed en masse into the mutation sorter (MS) strain and grown intwo separate cultures, one supplemented with tetracycline and the otherwith streptomycin. DNA from each of the two cultures was fluorescentlylabeled and loaded on the ABI sequencing machine. Fragment peaks wereanalyzed and the presence or absence of variations in a particular DNAfragment was assessed by determining the pool where a specific fragmentwas more prevalent (FIG. 7).

Shown in FIG. 7, the two samples prepared from the cultures supplementedwith streptomycin or tetracycline, respectively, have different peaktraces. The traces show the different peaks corresponding to thedifferent fragments. Each peak is quantitated automatically. Assignmentof the alleles of the tested individual is determined from the relativeintensity of a fragment between the two pools. The predominance of afragment in the streptomycin pool indicates the absence of a variation.In contrast, the predominance of a fragment in the tetracycline poolindicates the presence of a variation on both alleles. Finally, theabundance of a fragment in both pools indicates the individual tested isheterozygous for the fragment. These assignments are reproducible inindependent testing. The signal to noise ratio for detecting aheterozygous variation is 10:1; and the detection of homozygousvariation is substantially more robust.

The above experiment was performed on a nuclear family of 3 individuals.Each individual was tested three independent times. Variations weredetected in all three individuals. Robust signal to noise ratio wasobtained; heterozygous alleles were effectively identified with theaverage signal to noise ratio of 10:1. These results were perfectlyreproducible as exactly the same variant fragments were detected in anindividual in each of the three independent experiments. Differentfragments were variant in the different individuals; the pattern ofvariation among the three people followed a Mendelian mode ofinheritance. In these experiments the sensitivity, specificity andreproducibility of detecting DNA variations by MRD, as well as itspotential for high throughput variation screening have beendemonstrated.

MRD technology makes the large scale screening of candidate genes foridentifying variations that cause common diseases a real possibility. Atthe heart of this approach lies the ability to identify genomic sequencevariations that are more frequent in a particular patient group than thenormal population. Identifying these variations can influence manyaspects of modern medicine: determining diagnoses, assessing prognosesand devising treatments for human diseases. The knowledge of geneticfactors causing common disease will impact medical care in a similar wayit has already influenced care for rare simple Mendelian diseases.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO 1 <211> LENGTH: 34<212> TYPE: DNA <213> ORGANISM: lambda phage <400> SEQUENCE: 1ataacttcgt ataatgtatg ctatacgaag ttat        #                  #        34 <210> SEQ ID NO 2 <211> LENGTH: 34 <212> TYPE: DNA<213> ORGANISM: lambda phage <400> SEQUENCE: 2acaacttcgt ataatgtatg ctatacgaag ttat        #                  #        34

What is claimed is:
 1. A method of detecting a mismatch in any of aplurality of DNA duplexes of distinct nucleic acid sequence, saidduplexes formed in a single hybridization reaction, comprising:detecting, for any of said duplexes, an alteration in a bacterial cellcharacteristic, said alteration effected by the in vivo mismatchcorepair of at least 5 contiguous nucleotides of a marker that ispresent together with said duplex in a vector within said bacterialcell, said corepair being initiated by a mismatch of no more than 4contiguous nucleotides in said duplex.
 2. The method of claim 1, whereinsaid plurality includes duplexes of at least 10 distinct nucleic acidsequences.
 3. The method of claim 2, wherein said plurality includes atleast 100 duplexes of distinct nucleic acid sequence.
 4. The method ofclaim 3, wherein said plurality includes at least 10,000 duplexes ofdistinct nucleic acid sequence.
 5. The method of claim 3, wherein saidplurality includes at least 100,000 duplexes of distinct nucleic acidsequence.
 6. The method of claim 1, wherein said plurality includesnucleic add sequences derived from a prokaryote.
 7. The method of claim1, wherein said plurality includes nucleic acid sequences derived from avirus.
 8. The method of claim 1, wherein said plurality includes nucleicacid sequences derived from a eukaryote.
 9. The method of claim 8,wherein said eukaryote is a mammal.
 10. The method of claim 9, whereinsaid mammal is a human.
 11. The method of claim 10, wherein saidplurality includes nucleic acid sequences derived from the coding regionof a human gene.
 12. The method of claim 11, wherein said human gene isselected from the group consisting of: hemoglobin, dystrophin, BRCA1,BRCA2, CFTR, factor VIII, factor IX, oncogenes, tumor suppressors, andgenes on human chromosome
 21. 13. The method of claim 1, wherein saidmismatch in said duplex is a single nucleotide polymorphism.
 14. Themethod of claim 1, wherein said marker is inactivated by said in vivomismatch corepair.
 15. The method of claim 1, wherein said marker is arecombinase.
 16. The method of claim 15, wherein said recombinase is Crerecombinase.
 17. The method at claim 1, wherein said bacterial cellcharacteristic is selected from the group consisting of: cell color,luminescence, antibiotic sensitivity, and antibiotic resistance.
 18. Themethod of claim 15, wherein mismatch corepair of said recombinase alterssaid bacterial cell's antibiotic resistance or sensitivity.
 19. Themethod of claim 1, further comprising the antecedent step of formingsaid plurality of DNA duplexes by annealing first nucleic acid strandssaid first strands including at least one nucleic acid sequence, tosecond nucleic acid strands, said second strands including a pluralityof distinct nucleic acid sequences.
 20. The method of claim 19, whereinsaid plurality of second nucleic acid strands are derived from a commonsource.
 21. The method of claim 20, wherein said common source isgenomic DNA from a single individual.
 22. The method of claim 20,wherein said common source is cDNA from a single individual.
 23. Themethod of claim 19, wherein said plurality of second nucleic acidstrands are derived from a pooled source.
 24. The method of claim 23,wherein said source is pooled from family members.